A novel high-titer, bifunctional lentiviral vector for autologous hematopoietic stem cell gene therapy of sickle cell disease

A major limitation of gene therapy for sickle cell disease (SCD) is the availability and access to a potentially curative one-time treatment, due to high treatment costs. We have developed a high-titer bifunctional lentiviral vector (LVV) in a vector backbone that has reduced size, high vector yields, and efficient gene transfer to human CD34+ hematopoietic stem and progenitor cells (HSPCs). This LVV contains locus control region cores expressing an anti-sickling βAS3-globin gene and two microRNA-adapted short hairpin RNA simultaneously targeting BCL11A and ZNF410 transcripts to maximally induce fetal hemoglobin (HbF) expression. This LVV induces high levels of anti-sickling hemoglobins (HbAAS3 + HbF), while concurrently decreasing sickle hemoglobin (HbS). The decrease in HbS and increased anti-sickling hemoglobin impedes deoxygenated HbS polymerization and red blood cell sickling at low vector copy per cell in transduced SCD patient CD34+ cells differentiated into erythrocytes. The dual alterations in red cell hemoglobins ameliorated the SCD phenotype in the SCD Berkeley mouse model in vivo. With high titer and enhanced transduction of HSPC at a low multiplicity of infection, this LVV will increase the number of patient doses of vector from production lots to decrease costs and help improve accessibility to gene therapy for SCD.

The Townes SCD mouse model shows poor induction of HbF.
We performed a study to assess the activity of the UV1-SS and UV1-DS vectors in the Townes mouse SCD murine model, which reproduce several hematologic and pathophysiologic aspects of clinical disease. 1,2We first cloned fetal-globin positive control vectors that only express gammaglobin, by knocking out the β AS3 -globin open reading frame in the UV1-SS and UV1-DS vectors, creating UV1-SS (No AS3) and UV1-DS (No AS3).To generate the controls all methionine codons (ATG) in the β AS3 -globin open reading frame were mutated.Site-directed mutagenesis was used to change the ATG start codon as well as a downstream methionine codon in exon 2 (Figure S7A).The single or double shmiR s were left unchanged.
We validated the fetal globin positive controls by performing an erythroid differentiation using the parental HUDEP-2 cell line.The cells were transduced with vectors at 3.0x10 5 TU/mL without transduction enhancers to aim for a VCN of 1-2.The cells were differentiated into erythrocytes and were collected on day 7 for VCN analysis and on day 12 for protein.Protein was analyzed by HPLC (Figure S7B, S7C).The HPLC data shows that UV1-SS (No AS3) and UV1-DS (No AS3) do not express Hbβ AS3 , while HbF is induced, suggesting that β AS3 -globin has been knocked out and will be appropriate controls in the UV1 backbone.
The control vectors were packaged and concentrated virus was generated through ultracentrifugation.Titer was determined for raw and concentrated supernatant through HT-29 transduction (Figure S7D).A myeloid dose response with Lin-cells was performed to determine which concentration to use for each vector (UV1, UV1-SS, UV1-DS, UV1-SS (No AS3), UV1-DS (No AS3)) to obtain a VCN around 4 for Townes mouse studies Figure S7E).Lin-Townes mouse BM cells were pre-stimulated at 1x10 6 cells/mL and transduced 24 hours later.24 hours post transduction the cells were transplanted by retro-orbital injection into lethally irradiated B6 CD45.1 recipients.PB, BM, and spleen were collected at week 16 for analysis.
There was a small induction of HbF in red blood cells in mice treated with vectors containing the single or double shmiR s after intracellular staining of HbF and flow cytometry analysis (Figure S8B).However, there were not significant differences with the hematologic indices or spleen weights in untransduced SCD recipients (Figure S8C).These data demonstrate that the Townes mouse model does not induce HbF expression to a functional level when knocking down Bcl11a and Zfp410 with shmiR s , compared to the degree of HbF induction achieved in human HSPC.Due to minimal induction of HbF, we did not see correction of the erythroid manifestations of SCD in the Townes mouse model.Woodard et al. 3 observed a similar blunted induction of HbF in Townes mouse HSPC that were treated with CRISPR/Cas9 nuclease to disrupt gamma-globin gene promoter sites.

Figure S1 .
Figure S1.Schematic of experimental plan for the in vivo Berkeley SCD mouse model.Image created with BioRender.

Figure S2 .Figure S3 .Figure
Figure S2.Efficient knockdown of Zfp410 by Zfp410 shmiR vector leads to high Hbb-y induction in erythroid differentiated MEL cells in vitro.Zfp410 and Hbb-y mRNA expression as measured by RT-qPCR with Gapdh as control.Data represent mean ± SD.WT Zfp410 shmiR

Figure S7 .
Figure S7.Fetal globin positive control vector design, titer, and gene transfer.(A) Site-directed mutagenesis strategy to change methionine codons (ATG) in the β AS3 -globin open reading frame (B) HUDEP-2 parental cells were transduced with vectors at 3.0x10 5 TU/mL and then subjected to erythroid differentiation in vitro for 12 days and protein was assessed by HPLC.Induction of HbB AS3 (C) Induction of HbF (D) Vectors were packaged with an HEK293T PKR knock-out cell line and titers were determined by HT-29 cell line transduction, using raw viral supernatant, and quantified with ddPCR.Each point on the plot represents vector packaged and titered from an individual 10cm plate.(E)Lin-cells from Townes mice were transduced with constructs at 6x10 5 TU/mL, 2x10 6 TU/mL, 6x10 6 TU/mL and 2x10 7 TU/mL (MOI: 0.6, 2, 6, and 20) and cultured for 14 days under myeloid differentiation conditions to assess levels of infectivity.Vector copy number (VCN) was measured by ddPCR.Error bars represent mean ± SD.Image created with BioRender.

Figure S8 .
Figure S8.Peripheral blood sickle cell disease erythroid cell parameters in vivo in Townes SCD mouse model.Lineage negative (lin-) bone marrow cells from Townes mice (CD45.2) were transduced with each vector or mock-transduced as control and transplanted into irradiated B6 CD45.1+ (Pep Boy) mouse recipients.Mice were bled at 16 weeks after transplant and PB was analyzed.(A) Engraftment was assessed in PB by flow cytometry (%CD45.2+cells).(B) Percentage of fetal globin expressing cells was assessed by intracellular staining and flow cytometry.(C) Red blood cell counts (1x10 6 /uL) (D) Reticulocyte counts (%).(E) Hemoglobin (g/dL).(F) Hematocrit.Error bars represent mean ± SD.Each data point represents an individual mouse.

Figure S9 .
Figure S9.Vector Copy Number, HbF Expression by HPLC, and Spleen Weight Mice were euthanized at 16 weeks after transplant and whole bone marrow (BM) and spleen was harvested and analyzed individually.(A) VCN in BM was determined by ddPCR.(B) Percentages of HbF expression was determined by HPLC.(C) Spleen weights.Error bars represent mean ± SD.Each data point represents an individual mouse.